Sequencing of Tryptic Peptides Using Chemically Assisted Fragmentation and MALDI-PSD

  • John Flensburg
  • Maria Liminga
Part of the Springer Protocols Handbooks book series (SPH)


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become the preferred method for high-throughput identification of proteins using peptide mass fingerprinting (PMF), due to its ease of automation, short analysis time, relatively high tolerance towards contaminants, high sensitivity, and mass accuracy (1). In this technique, proteins are typically separated by twodimensional (2-D) gel electrophoresis, enzymatically in-gel digested with trypsin, extracted from the gel, and analyzed by MALDI-TOF MS. The resulting peptide mass fingerprints are compared to theoretical fingerprints from a protein- or DNA-sequence database for identification. In comparison to other ionization techniques such as electrospray ionization (ESI), the soft ionization induced by MALDI predominantly generates singly charged ions, which allows for a relatively easy interpretation of acquired spectra. Unfortunately, identification is not always unambiguous for a substantial fraction of the peptides analyzed, and it is not unusual that only a few peptides are recovered from an in-gel digest, especially when the protein is poorly expressed. To further improve the protein identification rate, amino acid sequence information from tryptic peptides is necessary. However, it is a well known fact that direct sequencing using MALDI post-source decay (PSD) often results in poor and unpredictable fragmentation patterns, which are mostly impossible to interpret (2). Singly charged tryptic peptides, formed during MALDI ionization, do not fragment readily because there is not enough internal energy available to move the ionizing proton from the basic C-terminal to the peptide backbone to induce fragmentation. In ESI, this problem is easily avoided by selecting doubly protonated peptides, which fragment readily.


Tryptic Peptide Peptide Mass Fingerprinting Unambiguous Identification Sulfonation Reaction Amino Acid Sequence Information 
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Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • John Flensburg
    • 1
  • Maria Liminga
    • 1
  1. 1.Amersham Biosciences AB, GE HealthcareUppsalaSweden

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